Malaria causes an estimated 500 million clinical cases and up to 2.7 million deaths per annum, reduces the GDP of Africa by >1% and is a serious risk for travelers including military personnel. Sanaria's goal is to develop and commercialize a >90% protective attenuated Plasmodium falciparum (Pf) sporozoite (SPZ) vaccine for two primary markets with potential annual revenues >$1 billion, travelers from the developed world and young children in the developing world. Immunization with radiation-attenuated (RA) PfSPZ can protect human volunteers for up to 10 months against Pf challenge. No other experimental malaria vaccine has given comparable protection. The development of a RA PfSPZ vaccine has to date been viewed as impractical because it was not considered feasible to immunize humans by a clinically acceptable parenteral route, produce adequate quantities of PfSPZ, and produce and characterize the aseptic, purified, stable, cryopreserved RA PfSPZ needed to meet regulatory and commercial standards for a vaccine. Sanaria has overcome all these problems, met with the FDA regarding plans for an Investigational New Drug application (IND) and will assess in humans the safety, immunogenicity and protective efficacy of its RA PfSPZ vaccine in 2008. A major consideration for routine production of large quantities of PfSPZ is the intensity of Pf infection in the mosquito;if the infection rates are increased, production is increased in direct proportion for no additional effort. Recent advances in a number of areas of mosquito molecular genetics provide Sanaria with unique opportunities to significantly increase PfSPZ production. Insect scientists have made considerable progress toward understanding how insects respond to infections by foreign microbes. In mosquitoes, three genes (TEP1, LRIM1, SRPN6) have recently been shown to play a major role in limiting the intensity of some Plasmodium infections. That is, these genes play roles in the mosquito's efforts to resist infection. Eliminating the expression of these genes results in insects that are more susceptible to infection. We propose to assess the specific role these genes play in the infection of An. stephensi by Pf and then create lines of An. stephensi in which the expression of some or all of these genes is eliminated. The mosquitoes resulting from such modifications will be extremely susceptible to Pf infection, yielding consistently higher numbers of sporozoites in the salivary glands. The three target genes will be assessed using established transient gene knockdown technologies in mosquitoes (double-stranded RNA injections into adult females). Based on those data, permanently modified mosquitoes will be created using established mosquito gene integration technologies. Modified mosquitoes will be assessed with respect to their Pf infection phenotype. The mosquitoes produced in this project will be prototypes that demonstrate the viability of this approach to enhancing sporozoite production. This project will lead to a phase II project in which new modified mosquitoes will be constructed that optimize temporal and spatial expression patterns of transgenes, incorporate fail-safe transgene stabilization features, eliminate foreign transgenes from the gene vector and incorporate a biosafety transgene that results in mosquitoes that can only be grown in the laboratory. These mosquitoes will be evaluated within the context of large-scale sporozoite production efforts. PUBLIC HEALTH RELEVANCE - PROJECT NARRATIVE: Malaria causes 500 million clinical cases and 1-3 million deaths annually, is responsible for >1% loss of GDP in Africa annually and is a serious concern for travelers and military personnel. Sanaria's goal is to develop and commercialize a >90% protective malaria vaccine for two primary markets with a potential for >$1 billion annual revenues: 1) Travelers from the developed world;2) Infants, young children, and adolescent girls in the developing world. Success in this project will significantly decrease the cost of development and production of the vaccine, and reduce the time to market for this malaria vaccine.